The transcription factor JunB

The transcription factor JunB belongs to the AP-1 family with the predicted size of ~35.7 kDa.

The expression of JunB can be triggered by numerous extracellular stimuli, such as serum, growth factors, phorbol esters (TPA) and activators of protein kinase A (PKA) due to transcriptional activation of the junB promoter. The 5´flanking region of junB contains recognition sequences for Smad and Ets, (Coffer et al., 1994), transcription factors, which can be regulated by MAP-kinases.

Further, it contains an IL-6 response element (IL-6RE) (Nakajima et al., 1993) containing a STAT3 binding site and a CRE and SRE (serum response element) like site (Kitabayashi et al., 1993), a GC rich region, an inverted repeat (IR) element (de Groot et al., 1991) and a myeloid-specific IL-6RE (Sjin et al., 1999) in the proximal promoter region. Additionally, the regulation of JunB by v-src involves the CAAT and TATA box region (Apel et al., 1992).

Introduction

51 AP-1 transcription factors integrate both mitogenic and stress signaling pathways. Signaling pathways mediated by growth factors induce JunB through a TRE, a SRE and two Ets-linked motifs located in a region around -1000 to - 2000 in the mouse JunB promoter (Phinney et al., 1996). In contrast, Pdgfb, serum, bFGF, phorbol ester and forskolin, can induce JunB expression by a SRE and a CRE site located in the 3’ flanking region of the junB gene (Perez-Albuerne et al., 1993). Moreover, four NF-κB binding sites located downstream of the gene have been shown to mediate transcriptional induction of JunB in response to oxygen deprivation (Schmidt et al., 2007) (Figure 12).

Figure 12: Schematic model of the junB promoter.

JunB has been described to act antagonistically to c-Jun in transcriptional regulation and is proposed to be a negative regulator of cell proliferation (Chiu et al., 1989; Schutte et al., 1989). During organogenesis in mouse, c-Jun and JunB have distinct tissue-specific roles in cell proliferation and differentiation during fetal development (Wilkinson et al., 1989). In comparison to c-Jun, JunB has a ten-fold decreased activity to activate AP-1-responsive genes containing single AP-1 binding sites to induce oncogenic transformation, due to a small number of amino acid changes between its DNA-binding and dimerization motifs (Deng and Karin, 1993). But strikingly, JunB appears to be as effective as Jun in trans-activating reporter genes containing multiple AP-1 binding sites which suggest, that trans-activation by JunB may require synergistic interactions between multiple homodimers bound to adjacent sites (Angel and Karin, 1991; Chiu et al., 1989).

Increased JunB expression can suppress cell proliferation by transcriptional activation of the cyclin-dependent kinase inhibitor p16^INK4α and elevated JunB expression in 3T3 cells also inhibited Ras- and Src-mediated transformation and tumor growth in vivo (Passegue and Wagner, 2000). Further, the antagonism between c-Jun and JunB plays an important role in regulating cell cycle progression, through negatively regulating cyclin D1 during mitosis by increasing c-Jun activating phosphorylation and triggering the phosphorylation dependent degradation of JunB via p34^cdc2-cyclin B kinase on three proline-flanked serine or threonine residues (Ser23, Thr150 and Ser186) in M and early G1 phase of the cell cycle (Bakiri et al., 2000).

52 Surprisingly, high JunB expression in S phase, drop during mid- to late G2 phase due to accelerated phosphorylation-dependent degradation by the proteasome, an incident which is absolutely required for proper mitosis. Consistently, abnormal JunB expression in late G2 phase entails a variety of mitotic defects, thus JunB might contribute to tumorigenesis (Farras et al., 2008). Moreover, JunB is suggested to become phosphorylated by JNK in T cells at threonine residues 102 and 104 which is important for synergy with c-Maf transcription factor to activate IL-4 expression and T helper cell differentiation (Li et al., 1999).

Beside, JNKs are responsible to regulate the E3 ubiquitin ligase Itch via phosphorylation dependent activation. Itch can bind via the WW domains to the proline-rich sequence PPxY (PY) motif of JunB which targets JunB for ubiquitination in T cells (Gao et al., 2004). Mice lacking Itch show an accumulation of JunB in helper T cells, which lead to an increased Th2 differentiation (Fang et al., 2002).

One mechanism by which AKT kinase-dependent hypersensitivity to mammalian target of rapamycin (mTOR) inhibitor is controlled, is by the differential expression of cyclin D1 and c-MYC. Vartanian et al. could recently show, that AKT-kinase dependent Itch-mediated JunB degradation has a regulatory function in the transcriptional responses of cyclin D1 and c-MYC to rapamycin (Vartanian et al., 2010). Furthermore, the (HECT)-type ubiquitin ligase Smurf1 interacts with JunB through the PPxY motif and negatively regulates mesenchymal stem cell (MSC) proliferation and differentiation by controlling JunB turnover through ubiquitination and the proteasome pathway. This represents another regulatory mechanism controlling JunB function in cells (Zhao et al., 2010).

Recently, Das et al. described that an increased expression of the disulfide oxidoreductase Thioredoxin (Trx), a protein involved in redox dependent cellular functions, activates NFκB and induces enhanced binding and transactivation potential of the AP-1 family members, JunB, c-Jun and Fra1, by activating JNK subgroup of MAPKs (Das, 2001; Das and Muniyappa, 2010).

JunB has a unique, non-redundant function in vivo, because it is required for proper endothelial morphogenesis (Licht et al., 2006) and lack of JunB in mice causes defects in placentagenesis and vascularisation which leads to embryonic lethality between day 8.5 and 10.0 (Schorpp-Kistner et al., 1999). Moreover, it was shown, that loss of JunB expression in the epidermis of adult mice severely affects the skin and bone formation due to high systemic levels of the negatively regulated JunB-target granulocyte colony-stimulating factor (G-CSF) (Hess et al., 2003; Meixner et al., 2008).

Introduction

53 Interestingly, JunB loss in mice has an impact on fat metabolism, due to increased level of adipose triglyceride lipase and hormone-sensitive lipase, the key enzymes of lipolysis (Pinent et al., 2011). Further, JunB represses the IL-6 promoter, which is reflected in the fact, that epidermal loss of JunB leads to a Systemic lupus erythematosus (SLE) phenotype, a complex autoimmune disease, due to hyper IL-6 signaling (Pflegerl et al., 2009). Beside, JunB is an essential target gene of hypoxia-induced signaling via NF-κB, responsible for hypoxia-mediated vascular endothelial growth factor (VEGF) expression and tumor angiogenesis, which is hypoxia-inducible factor (HIF) independent (Schmidt et al., 2007;

Textor et al., 2006; Wang et al., 2010). Thus, JunB is a critical independent regulator of the autocrine and paracrine acting VEGFα. Interestingly, a decreased JunB expression was further described for synovial fibroblasts from rheumatoid arthritis patients (Huber et al., 2003). Moreover, deletion leads to a phenotype resembling the histological and molecular hallmarks of psoriasis, including arthritic lesions (Zenz et al., 2005).

JunB has revealed an important regulatory role in the hematopoietic system and tumorigenesis, since JunB inactivation in postnatal mice results in a myeloproliferative disease resembling early human chronic myelogenous leukemia (CML) (Passegue et al., 2001; Passegue et al., 2004). In agreement with this, JunB expression is diminished in some human chronic myeloid leukemia (Yang et al., 2003) and B cell leukemias (Ott et al., 2007;

Szremska et al., 2003), and downregulation of JunB partially depends on junB gene methylation (Yang et al., 2003).

JunB is also described as a major determinant for maintenance of dividing and adult muscle cells by inducing hypertrophy and thereby efficiently preventing atrophy (Raffaello et al., 2010). Another emergent role for JunB was characterized for the differentiation of naïve CD4+ T cells into T helper1 and 2 cells, which are classified by their specific set of cytokines.

Here, JunB expression is not only essential, but has to be tightly adjusted to ensure a proper cell function, such as a Th2 immune response (Hartenstein et al., 2002).

Furthermore, the post-translational modification of JunB with small ubiquitin-like modifier (SUMO) on lysine 237 plays a critical role in T cell activation. Indeed, SUMOylation of JunB regulates its ability to induce cytokine gene transcription and likely plays a critical role in T cell activation (Garaude et al., 2008). For natural killer (NK) cell activation, JunB plays a pivotal role in the negative regulation of the NKG2D-ligand, activating receptor RAE-1-epsilon expression (Nausch et al., 2006). Moreover, Textor et al. described novel functions for JunB in regulating novel target genes in mast cells, which are required for proper mast cell degranulation and mast cell mediated angiogenic processes (Textor et al., 2007). Very recently, another level of regulation is provided where JunB and c-Rel cooperatively enhance Foxp3 expression during induced regulatory T (iTreg) cell differentiation (Son et al., 2011).

54 A further level of regulation implicates the AP-1 proteins with autophagy. Interestingly, unlike c-Jun, which can enhance apoptosis, the data implicating JunB in the regulation of apoptosis are less significant. In fact, it has been suggested that JunB inhibits cytokine-mediated apoptosis in an insulin-producing cell line and in purified rat primary beta cells (Gurzov et al., 2008), or in nigral neurons following axotomy (Winter et al., 2002). Conversely, Yogev et al.

could show recently, that JunB and c-Jun can inhibit autophagy induced by starvation, overexpression of a short form of ARF (smARF) or after rapamycin treatment and deregulation of JunB expression, when autophagy is specifically required, tilts the fate of starved cells to apoptosis (Yogev et al., 2010; Yogev and Shaulian, 2010).

Although JunB has been considered to be a tumor suppressor, indeed, it has also a cell cycle-promoting role through activating the transcription of the cell cycle regulator cyclin A (Andrecht et al., 2002). Passegue et al. demonstrated, using a knock-in strategy and a transgenic complementation approach, that JunB may substitute for c-Jun in improving survival of c-Jun-deficient embryos (Passegue et al., 2002). These data support previous studies which demonstrated that JunB is required for cell cycle re-entry after quiescence (Kovary and Bravo, 1991) and can cooperate with c-Jun in the development of mouse fibrosarcoma (Bossy-Wetzel et al., 1992), and collectively suggest that JunB is able to enhance proliferation, cooperate with c-Jun and promote neoplastic transformation, especially lymphomas. This goes along with findings revealing that in some hyperproliferative T cell lymphomas JunB was highly overexpressed (Mao et al., 2004; Rassidakis et al., 2005).

Accordingly, JunB is a critical regulator of the expression of cytokines important for T lymphocyte proliferation and differentiation, namely IL-4 (Hartenstein et al., 2002) and IL-2 (Jain et al., 1992; Li et al., 1999). Beside, JunB expression and binding capacity are decreased in anergic T cells, which do not produce IL-2 (Heisel and Keown, 2001; Mondino et al., 1996; Schwartz, 1997; Sundstedt and Dohlsten, 1998).

Moreover, aberrant JunB and CD30 expression is involved in the development of Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL), whereby initiation and constitutive transcription of CD30 gene by aberrant JunB expression maintains hypomethylation of CD30 CpG islands, contributing to the pathogenesis of HL and ALCL (Watanabe et al., 2008).

In addition, JunB, like c-Jun, binds to the promoter of the tumor promoter Dmp1 and elevates its expression. Dmp1 subsequently up-regulates the expression of p19ARF, another tumor suppressor residing in the INK4 locus, which is a major regulator of p53 activity (Sreeramaneni et al., 2005). These mechanisms may also be the basis for the enhancement of drug-induced senescence by JunB (Yogev et al., 2006).

Aim of this study

55